Targeting SIGLEC15 as an emerging immunotherapy for anaplastic thyroid cancer.

Anaplastic thyroid carcinoma (ATC) is the most aggressive subtype of thyroid cancer with few effective therapies. Though immunotherapies such as targeting PD-1/PD-L1 axis have benefited patients with solid tumor, the druggable immune checkpoints are quite limited in ATC. In our study, we focused on the anti-tumor potential of sialic acid-binding Ig-like lectins (Siglecs) in ATC. Through screening by integrating microarray datasets including 216 thyroid-cancer tissues and single-cell RNA-sequencing, SIGLEC family members CD33, SIGLEC1, SIGLEC10 and SIGLEC15 were significantly overexpressed in ATC, among which SIGLEC15 increased highest and mainly expressed on cancer cells. SIGLEC15high ATC cells are characterized by high expression of serine protease PRSS23 and cancer stem cell marker CD44. Compared with SIGLEC15low cancer cells, SIGLEC15high ATC cells exhibited higher interaction frequency with tumor microenvironment cells. Further study showed that SIGLEC15high cancer cells mainly interacted with T cells by immunosuppressive signals such as MIF-TNFRSF14 and CXCL12-CXCR4. Notably, treatment of anti-SIGLEC15 antibody profoundly increased the cytotoxic ability of CD8+ T cells in a co-culture model and zebrafish-derived ATC xenografts. Consistently, administration of anti-SIGLEC15 antibody significantly inhibited tumor growth and prolonged mouse survival in an immunocompetent model of murine ATC, which was associated with increase of M1/M2, natural killer (NK) cells and CD8+ T cells, and decrease of myeloid-derived suppressor cells (MDSCs). SIGLEC15 inhibited T cell activation by reducing NFAT1, NFAT2, and NF-κB signals. Blocking SIGLEC15 increased the secretion of IFN-γ and IL-2 in vitro and in vivo. In conclusion, our finding demonstrates that SIGLEC15 is an emerging and promising target for immunotherapy in ATC.

A zebrafish xenotransplant model of anaplastic thyroid cancer to study tumor microenvironment and innate immune cell interactions in vivo.

Anaplastic thyroid cancer (ATC) is of the most aggressive thyroid cancer. While ATC is rare, it accounts for a disproportionately high number of thyroid cancer-related deaths. Here, we developed an ATC xenotransplant model in zebrafish larvae, where we can study tumorigenesis and therapeutic response in vivo. Using both mouse (T4888M) and human (C643)-derived fluorescently labeled ATC cell lines, we show these cell lines display different engraftment rates, mass volume, proliferation, cell death, angiogenic potential, and neutrophil and macrophage recruitment and infiltration. Next, using a PIP-FUCCI reporter to track proliferation in vivo, we observed cells in each phase of the cell cycle. Additionally, we performed long-term non-invasive intravital microscopy over 48 h to understand cellular dynamics in the tumor microenvironment at the single-cell level. Lastly, we tested two drug treatments, AZD2014 and a combination therapy of dabrafenib and trametinib, to show our model could be used as an effective screening platform for new therapeutic compounds for ATC. Altogether, we show that zebrafish xenotransplants make a great model to study thyroid carcinogenesis and the tumor microenvironment, while also being a suitable model to test new therapeutics in vivo.

A distinct tumor microenvironment makes anaplastic thyroid cancer more lethal but immunotherapy sensitive than papillary thyroid cancer.

Both anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) originate from thyroid follicular epithelial cells, but ATC has a significantly worse prognosis and shows resistance to conventional therapies. However, clinical trials found that immunotherapy works better in ATC than late-stage PTC. Here, we used single-cell RNA sequencing (scRNA-Seq) to generate a single-cell atlas of thyroid cancer. Differences in ATC and PTC tumor microenvironment components (including malignant cells, stromal cells, and immune cells) leading to the polarized prognoses were identified. Intriguingly, we found that CXCL13+ T lymphocytes were enriched in ATC samples and might promote the development of early tertiary lymphoid structure (TLS). Last, murine experiments and scRNA-Seq analysis of a treated patient's tumor demonstrated that famitinib plus anti-PD-1 antibody could advance TLS in thyroid cancer. We displayed the cellular landscape of ATC and PTC, finding that CXCL13+ T cells and early TLS might make ATC more sensitive to immunotherapy.

Evaluation of E-Cadherin and ß-Catenin Immunoreactivity for Determining Undifferentiated Cells in Anaplastic Thyroid Carcinoma.

INTRODUCTION: An immunohistochemical study has occasionally been performed to diagnose anaplastic thyroid carcinoma (ATC). However, antibodies to confirm the undifferentiated nature of ATC have not yet been evaluated. The aim of this study was to evaluate E-cadherin and ß-catenin expressions in immunoreactivity to determine undifferentiated carcinoma cells in the diagnosis of ATC. METHODS: We immunohistochemically examined 29 ATCs, 30 poorly differentiated thyroid carcinomas (PDTCs), 22 well-differentiated thyroid carcinomas (WDTCs), and 3 squamous cell carcinomas. Antibodies for thyroid transcription factor-1 (TTF-1), paired-box gene 8 (PAX8), ß-catenin, and E-cadherin were used. RESULTS: All WDTCs tested positive for TTF-1, PAX8, and E-cadherin. The positive rates of TTF-1, PAX8, and E-cadherin were 93.3, 93.3, and 100%, respectively, in PDTCs and 17.2, 51.7, and 10.3%, respectively, in ATCs. WDTC expressed the lateral cell membrane staining for ß-catenin and E-cadherin, whereas PDTC showed circumferential cell membranous expression (fishnet pattern). ß-catenin cell membrane expression in ATCs is lost or discontinuous. Carcinoma cells with ß-catenin nuclear expression without cell membranous expression were scattered in 72.4% of ATCs but were not observed in the other carcinomas. CONCLUSION: We propose 3 immunohistochemical findings to determine undifferentiated carcinoma cells in the diagnosis of ATC: (1) ß-catenin nuclear expression with no or reduced cell membranous expression, (2) the loss or discontinuous pattern of E-cadherin expression, and (3) the loss of PAX8 nuclear expression.

M2­like tumour­associated macrophage­secreted IGF promotes thyroid cancer stemness and metastasis by activating the PI3K/AKT/mTOR pathway.

M2­like tumour­associated macrophages (TAMs) have been demonstrated to promote the growth of anaplastic thyroid carcinoma (ATC). However, the underlying mechanism of M2­like TAMs in ATC remains unclear. Thus, in the present study, the role and mechanism of M2­like TAMs in ATC were investigated. M2­like TAMs were induced by treatment with PMA, plus IL­4 and IL­13, and identified by flow cytometry. Transwell and sphere formation assays were applied to assess the invasion and stemness of ATC cells. The expression levels of insulin­like growth factor (IGF)­1 and IGF­2 were examined by ELISA and reverse transcription­quantitative PCR. Proteins related to the epithelial­mesenchymal transition (EMT), stemness and the PI3K/AKT/mTOR pathway were examined via western blotting. Immunohistochemistry (IHC) was used to detect the expression of the M2­like TAM markers CD68 and CD206 in ATC tissues and thyroid adenoma tissues. It was found that treatment with PMA plus IL­4 and IL­13 successfully induced M2­like TAMs. Following co­culture with M2­like TAMs, the invasive ability and stemness of ATC cells were significantly increased. The expression levels of the EMT­related markers N­cadherin and Vimentin, the stemness­related markers Oct4, Sox2 and CD133, and the insulin receptor (IR)­A/IGF1 receptor (IGF1R) were markedly upregulated, whereas E­cadherin expression was significantly decreased. In addition, the production of IGF­1 and IGF­2 was significantly increased. Of note, exogenous IGF­1/IGF­2 promoted the invasion and stemness of C643 cells, whereas blocking IGF­1 and IGF­2 inhibited metastasis and stemness by repressing IR­A/IGF­1R­mediated PI3K/AKT/mTOR signalling in the co­culture system. IHC results showed that the expression of CD68 and CD206 was obviously increased in ATC tissues. To conclude, M2­like TAMs accelerated the metastasis and increased the stemness of ATC cells, and the underlying mechanism may be related to the section of IGF by M2­like TAMs, which activates the IR­A/IGF1R­mediated PI3K/AKT/mTOR signalling pathway.

CSPG4 Is a Potential Therapeutic Target in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is a rare cancer with poor prognosis and few treatment options. The objective of this study was to investigate new immune-associated therapeutic targets by identifying ATC-derived, human leukocyte antigen (HLA) class II-presenting peptides. One protein that generated multiple peptides in ATC was chondroitin sulfate-proteoglycan-4 (CSPG4), a transmembrane proteoglycan with increased expression in multiple aggressive cancers, but not yet investigated in ATC. Methods: We applied autologous peripheral blood T cells to ATC patient-derived xenografted mice to examine whether ATC induces a tumor-specific T cell response. We then identified peptide antigens eluted from the HLA-DQ complex in ATC patient-derived cells using mass spectrometry, detecting abundant CSPG4-derived peptides specific to the ATC sample. Next, we analyzed the surface expression level of CSPG4 in thyroid cancer cell lines and primary cell culture using flow cytometry. In addition, we used immunohistochemistry to compare the expression level and localization of the CSPG4 protein in ATC, papillary thyroid cancer, and normal thyroid tissue. We then investigated the correlation between CSPG4 expression and clinicopathological features of patients with thyroid cancer. Results: We found that ATC tissue had a high level of HLA-DQ expression and that the patient's CD4+ T cells showed activation when exposed to ATC. By eluting the HLA-DQ complex of ATC tissue, we found that CSPG4 generated one of the most abundant and specific peptides. CSPG4 expression at the cell surface of thyroid cancer was also significantly high when determined by flow cytometry, with the majority of ATC cell lines exhibiting ∼10-fold higher mean fluorescence intensity. Furthermore, most ATC patient cases expressed CSPG4 in the cytoplasm or membrane of the tumor cells. CSPG4 expression was correlated with tumor size, extrathyroidal extension, and intercellular adhesion molecule-1 (ICAM-1) circumferential expression. CSPG4 mRNA overexpression was associated with worse overall survival in patients with ATC and poorly differentiated thyroid cancer. Conclusions: CSPG4 expression is significantly elevated in aggressive thyroid cancers, with a strong correlation with a poor prognosis. The vast number of HLA-DQ eluted CSPG4 peptides was identified in ATC, demonstrating the potential of CSPG4 as a novel immunotherapeutic target for ATC.

LncRNA UCA1 attenuated the killing effect of cytotoxic CD8 + T cells on anaplastic thyroid carcinoma via miR-148a/PD-L1 pathway.

BACKGROUND: LncRNAs play an important role in the regulation of the killing effect of cytotoxic CD8 + T cells in various cancers. However, the role and underlying mechanisms of UCA1 in the killing effect of cytotoxic CD8 + T cells in anaplastic thyroid carcinoma (ATC) are not clear. METHODS: UCA1, miR-148a, and PD-L1 expression were detected by quantitative real-time PCR and/or Western blot. The ratio of PD-L1+ATC cells/ATC cells was determined using flow cytometry. The ability of CD8 + T cells to kill target ATC cells was detected by Chromium-51 (51Cr) release assay. The targeted relationship between UCA1 and miR-148a was confirmed by dual-luciferase reporter gene assay. RESULTS: UCA1 and PD-L1 expression levels were elevated in ATC tissues and cells. Silencing UCA1 and PD-L1 enhanced the killing effect of cytotoxic CD8 + T cells on ATC cells. UCA1 negatively regulated the expression of miR-148a, and miR-148a targeted PD-L1 to down-regulate its expression. Besides, we found that UCA1 attenuated the killing effect of cytotoxic CD8 + T cells and reduced cytokine secretion through PD-L1 and miR-148a. Finally, silencing UCA1 or PD-L1 in ATC cells restored the suppression of the killing effect of CD8 + T cells in vivo. CONCLUSION: UCA1 attenuated the killing effect of cytotoxic CD8 + T cells on ATC cells through the miR-148a/PD-L1 pathway.

Immune Profiling of Advanced Thyroid Cancers Using Fluorescent Multiplex Immunohistochemistry.

Background: Advanced thyroid cancers, including differentiated thyroid carcinoma (DTC) with distant metastasis, and anaplastic thyroid carcinoma (ATC), are associated with poor clinical outcomes and limited treatment options. This study aimed to determine the immune profiles of advanced thyroid cancers using fluorescent multiplex immunohistochemistry (F-MIHC) and multispectral imaging (MSI). Methods: Twenty-eight tissue samples were collected from 12 patients who had DTC with distant metastasis and from 16 with ATC. The samples were assessed using F-MIHC and MSI with antibodies against the cell surface molecules, cluster of differentiation (CD)4, CD8, programmed cell death-1 (PD-1), PD ligand 1 (PD-L1), forkhead box protein 3, and cytokeratin (CK). The expression of PD-L1 was evaluated using tumor proportion score (TPS) and combined positive score (CPS). Results: Significantly, more PD-L1-positive tumor cells (CK+PD-L1+) per mm2 were found in ATC samples than in DTC samples (183.5 vs. 0.03, p < 0.001). Lymphocyte infiltration was significantly increased in ATC compared with DTC, with significantly more PD-L1- or PD-1-positive lymphocytes in ATC samples than in DTC samples. The TPS and CPS for PD-L1 expression were negative in all DTC samples but positive in 81% and 94% of ATC samples, respectively. Conclusions: Immune profiling revealed significant differences between advanced DTC and ATC, particularly in terms of PD-L1 expression and lymphocyte infiltration. Therefore, immune profiling using F-MIHC and MSI can provide invaluable information regarding tumor microenvironments, which could help select candidates for immunotherapy.

Detecting Immune Response to Therapies Targeting PDL1 and BRAF by Using Ferumoxytol MRI and Macrin in Anaplastic Thyroid Cancer.

Background Anaplastic thyroid cancer (ATC) is aggressive with a poor prognosis, partly because of the immunosuppressive microenvironment created by tumor-associated macrophages (TAMs). Purpose To understand the relationship between TAM infiltration, tumor vascularization, and corresponding drug delivery by using ferumoxytol-enhanced MRI and macrin in an ATC mouse model. Materials and Methods ATC tumors were generated in 6-8-week-old female B6129SF1/J mice through intrathyroid injection to model orthotopic tumors, or intravenously to model hematogenous metastasis, and prospectively enrolled randomly into treatment cohorts (n = 94 total; August 1, 2018, to January 15, 2020). Mice were treated with vehicle or combined serine/threonine-protein kinase B-Raf (BRAF) kinase inhibitor (BRAFi) and anti-PDL1 antibody (aPDL1). A subset was cotreated with therapies, including an approximately 70-nm model drug delivery nanoparticle (DDNP) to target TAM, and an antibody-neutralizing colony stimulating factor 1 receptor (CSF1R). Imaging was performed at the macroscopic level with ferumoxytol-MRI and microscopically with macrin. Genetically engineered BrafV600E/WT p53-null allografts were used and complemented by a GFP-transgenic derivative and human xenografts. Tumor-bearing organs were processed by using tissue clearing and imaged with confocal microscopy and MRI. Two-tailed Wilcoxon tests were used for comparison (≥five per group). Results TAM levels were higher in orthotopic thyroid tumors compared with pulmonary metastatic lesions by 79% ± 23 (standard deviation; P < .001). These findings were concordant with ferumoxytol MRI, which showed 136% ± 88 higher uptake in thyroid lesions (P = .02) compared with lung lesions. BRAFi and aPDL1 combination therapy resulted in higher tumor DDNP delivery by 39% ± 14 in pulmonary lesions (P = .004). Compared with the untreated group, tumors following BRAFi, aPDL1, and CSF1R-blocking antibody combination therapy did not show greater levels of TAM or DDNP (P = .82). Conclusion In a mouse model of anaplastic thyroid cancer, ferumoxytol MRI showed 136% ± 88 greater uptake in orthotopic thyroid tumors compared with pulmonary lesions, which reflected high vascularization and greater tumor-associated macrophage (TAM) levels. Serine/threonine-protein kinase B-Raf inhibitor and anti-programmed death ligand 1 antibody elicited higher local TAM levels and 43% ± 20 greater therapeutic nanoparticle delivery but not higher vascularization in pulmonary tumors. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Luker in this issue.

PD1 Blockade Enhances ICAM1-Directed CAR T Therapeutic Efficacy in Advanced Thyroid Cancer.

PURPOSE: Advanced thyroid cancers, including poorly differentiated and anaplastic thyroid cancer (ATC), are lethal malignancies with limited treatment options. The majority of patients with ATC have responded poorly to programmed death 1 (PD1) blockade in early clinical trials. There is a need to explore new treatment options. EXPERIMENTAL DESIGN: We examined the expression of PD-L1 (a ligand of PD1) and intercellular adhesion molecule 1 (ICAM1) in thyroid tumors and ATC cell lines, and investigated the PD1 expression level in peripheral T cells of patients with thyroid cancer. Next, we studied the tumor-targeting efficacy and T-cell dynamics of monotherapy and combination treatments of ICAM1-targeting chimeric antigen receptor (CAR) T cells and anti-PD1 antibody in a xenograft model of ATC. RESULTS: Advanced thyroid cancers were associated with increased expression of both ICAM1 and PD-L1 in tumors, and elevated PD1 expression in CD8+ T cells of circulating blood. The expression of ICAM1 and PD-L1 in ATC lines was regulated by the IFNγ-JAK2 signaling pathway. ICAM1-targeted CAR T cells, produced from either healthy donor or patient T cells, in combination with PD1 blockade demonstrated an improved ability to eradicate ICAM1-expressing target tumor cells compared with CAR T treatment alone. PD1 blockade facilitated clearance of PD-L1 high tumor colonies and curtailed excessive CAR T expansion, resulting in rapid tumor clearance and prolonged survival in a mouse model. CONCLUSIONS: Targeting two IFNγ-inducible, tumor-associated antigens-ICAM1 and PD-L1-in a complementary manner might be an effective treatment strategy to control advanced thyroid cancers in vivo.

Diagnostic and prognostic value of preoperative systemic inflammatory markers in anaplastic thyroid cancer.

BACKGROUND: Easily accessible, generalized, and inexpensive methods are expected to differentiate anaplastic thyroid carcinoma (ATC) from advanced differentiated thyroid cancer (aDTC). We aimed to explore potential diagnostic and prognostic value of systematic inflammatory markers (SIMs) in ATC and aDTC. METHODS: About 22 ATC, 101 aDTC, and 100 matched early DTC patients were analyzed retrospectively. SIMs included the comprehensive index, neutrophil-monocyte-platelet-to-lymphocyte ratio (NMPLR) and the previously reported ones. Receiver operating characteristic, Kaplan-Meier, and COX regression analyses were mainly conducted. RESULTS: NMPLR exhibited the highest area under the curve value 0.806 (P < .0001) to diagnose ATC from aDTC. NMPLR was identified as an independent risk factor for overall survival (OS) (hazard ratio [HR]: 47.821, 95% confidence interval [CI], 2.863-798.765, P = .007) in ATC, as well as for OS (HR: 7.360, 95% CI, 1.620-33.430, P = .010) and recurrence-free survival (HR: 4.172, 95% CI, 1.139-15.286, P = .031) in aDTC. Taken both refractory types (ATC and aDTC) together, NMPLR could independently predict OS (HR: 6.470; 95% CI, 2.134-19.616; P = .001). CONCLUSION: NMPLR is a generalized index. It showed excellent potential in differential diagnosis and survival prediction in refractory thyroid cancer. However, it needs to be validated in larger cohort and clinical practice.

PD-1 Blockade in Anaplastic Thyroid Carcinoma.

PURPOSE: Anaplastic thyroid carcinoma is an aggressive malignancy that is almost always fatal and lacks effective systemic treatment options for patients with BRAF-wild type disease. As part of a phase I/II study in patients with advanced/metastatic solid tumors, patients with anaplastic thyroid carcinoma were treated with spartalizumab, a humanized monoclonal antibody against the programmed death-1 (PD-1) receptor. METHODS: We enrolled patients with locally advanced and/or metastatic anaplastic thyroid carcinoma in a phase II cohort of the study. Patients received 400 mg spartalizumab intravenously, once every 4 weeks. The overall response rate was determined according to RECIST v1.1. RESULTS: Forty-two patients were enrolled. Adverse events were consistent with those previously observed with PD-1 blockade. Most common treatment-related adverse events were diarrhea (12%), pruritus (12%), fatigue (7%), and pyrexia (7%). The overall response rate was 19%, including three patients with a complete response and five with a partial response. Most patients had baseline tumor biopsies positive for PD-L1 expression (n = 28/40 evaluable), and response rates were higher in PD-L1-positive (8/28; 29%) versus PD-L1-negative (0/12; 0%) patients. The highest rate of response was observed in the subset of patients with PD-L1 ≥ 50% (6/17; 35%). Responses were seen in both BRAF-nonmutant and BRAF-mutant patients and were durable, with a 1-year survival of 52.1% in the PD-L1-positive population. CONCLUSION: To our knowledge, this is the first clinical trial to show responsiveness of anaplastic thyroid carcinoma to PD-1 blockade.

Dissecting Anaplastic Thyroid Carcinoma: A Comprehensive Clinical, Histologic, Immunophenotypic, and Molecular Study of 360 Cases.

Background: Anaplastic thyroid carcinoma (ATC) is nearly always fatal. Large studies on ATC are exceedingly rare. We aimed to study the clinical, genotypic, and histologic characteristics of ATC in the largest retrospective cohort of ATC to date. Methods: Three hundred sixty patients with ATC from two tertiary centers were studied. Molecular testing was performed in 126 cases including 107 using next-generation sequencing. Results: The median patients' age was 68 years. Differentiated thyroid carcinoma (DTC) was present in 208 cases (58%), the most common being papillary carcinoma (n = 150). The 1-, 2-, 3-, and 5-year overall survival (OS) was 36%, 17%, 13%, and 11%, respectively. On univariate analysis, age, resectability, chemotherapy, radiotherapy, margin status, encapsulation, gross residual disease, gross extrathyroidal extension, percentage, and size of ATC in the primary tumor predicted OS (p < 0.05). Age, resectability, chemotherapy, and gross residual disease were independent prognostic factors in the entire cohort, while gross residual disease was the only independent predictor of OS in patients who had resection of their tumor. BRAF, RAS, TERT promoter, TP53, PIK3CA, E1F1AX, and PTEN mutations were detected in 45%, 24%, 75%, 63%, 18%, 14%, and 14% of ATC, respectively. Concomitant BRAF/RAS and TERT mutations were associated with worse outcome than mutation in only one of the genes. BRAF-mutated and RAS-mutated ATCs had similar frequency of nodal and distant metastasis. Twelve cases were pure squamous cell carcinoma, 60% of which carried BRAFV600E mutation and showed a similar OS to other ATCs. Conclusions: (i) Gross residual disease remains the most crucial indicator of outcome in ATC. (ii) Encapsulation, margin status, percentage, and size of ATC in the primary were prognostically relevant. (iii) Pure thyroid squamous cell carcinoma may be considered as ATC given a BRAFV600E genotype and similar outcome. (iv) In contrast to DTC, BRAF-mutated and RAS-mutated ATCs have similar metastatic spread. (v) Concomitant mutations of BRAF or RAS with TERT confer a worse prognosis.

Anaplastic Thyroid Cancer Cells Induce the Release of Mitochondrial Extracellular DNA Traps by Viable Neutrophils.

Neutrophils are key effector cells that orchestrate inflammatory responses in the tumor microenvironment. Although neutrophil extracellular DNA traps (NETs) entrap and kill pathogens, they also contribute to chronic inflammation and cancer progression. Thyroid cancer (TC) is the most frequently occurring cancer of the endocrine system, accounting for 70% of deaths due to endocrine tumors. Although anaplastic TC (ATC) is rare among TCs, it is highly lethal. We demonstrated in a recent study that tumor-infiltrating neutrophil density correlated with TC size. Moreover, TC-derived soluble mediators modulate the human neutrophil phenotype. Our study aimed to investigate the involvement of NETs in human TC. Highly purified neutrophils from healthy donors were primed in vitro with a papillary TC or ATC cell line conditioned medium (CM) or with a normal thyroid CM as control. NET release was quantified using a High-Content Imaging System. Neutrophil viability was assessed by flow cytometry. Fluorescence microscopy, flow cytometry, and PCR were performed to determine the mitochondrial origin of ATC-induced NETs. ATC CM-primed neutrophils were cocultured with ATC cells to determine the effects exerted by NETs on cell proliferation. ATC CM induce NET release, whereas papillary TC or normal thyroid CM did not. ATC CM-induced NET production occurred in a reactive oxygen species-dependent and cell death-independent manner and was associated with mitochondrial reactive oxygen species production; the NETs contained mitochondrial DNA. ATC CM-primed neutrophils promoted ATC cell proliferation in a NET-dependent manner.

Increased cytoplasmatic expression of cancer immune surveillance receptor CD1d in anaplastic thyroid carcinomas.

BACKGROUND: Anaplastic thyroid carcinomas are associated with rapid tumor growth, short survival time and without any promising therapy to improve the poor prognosis. In this study, expression of immunoregulative receptor CD1d and lymphocyte infiltration in different thyroid tumors as well as in healthy tissue were analyzed in order to find new targets for an immunotherapeutic approach. METHODS: CD1d immunohistochemistry was performed in samples of 18 anaplastic, 17 follicular, 27 papillary, and 4 medullary thyroid carcinomas as well as in 19 specimens from normal thyroid tissue and additionally in 10 samples of sarcoma, seven malignant melanoma and three spindle-cell lung carcinoma. Furthermore, thyroid samples were stained with antibodies against CD3, CD20, CD56, CD68, and LCA in order to analyze lymphocyte infiltration. RESULTS: For the first time CD1d receptor expression on normal thyroid tissue could be demonstrated. Moreover, anaplastic thyroid carcinomas showed significantly higher expression levels compared to other thyroid samples. Most astonishingly, CD1d expression disappeared from the cellular surface and was detected rather in the cytoplasm of anaplastic thyroid carcinoma cells. In addition, histologically similar tumors to anaplastic carcinoma like sarcoma and malignant melanoma revealed distinct CD1d staining patterns. Furthermore, infiltration of T cells, B cells, and macrophages in anaplastic thyroid carcinomas was different when compared to normal thyroid tissue and all other thyroid carcinomas. CONCLUSIONS: Anaplastic thyroid carcinomas show significantly higher expression of CD1d, a receptor for NKT cells, which are subject of several anticancer therapy studies. These results may offer a novel approach to explore immunotherapeutic treatment options.

Targeting CD47 in Anaplastic Thyroid Carcinoma Enhances Tumor Phagocytosis by Macrophages and Is a Promising Therapeutic Strategy.

Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers, with a median survival of only three to six months. Standard treatment options and even targeted therapies have so far failed to improve long-term overall survival. Thus, novel treatment modalities for ATC, such as immunotherapy, are urgently needed. CD47 is a "don't eat me" signal, which prevents cancer cells from phagocytosis by binding to signal regulatory protein alpha on macrophages. So far, the role of macrophages and the CD47-signal regulatory protein alpha signaling axis in ATC is not well understood. Methods: This study analyzed 19 primary human ATCs for macrophage markers, CD47 expression, and immune checkpoints by immunohistochemistry. ATC cell lines and a fresh ATC sample were assessed by flow cytometry for CD47 expression and macrophage infiltration, respectively. CD47 was blocked in phagocytosis assays of co-cultured macrophages and ATC cell lines. Anti-CD47 antibody treatment was administered to ATC cell line xenotransplanted immunocompromised mice, as well as to tamoxifen-induced ATC double-transgenic mice. Results: Human ATC samples were heavily infiltrated by CD68- and CD163-expressing tumor-associated macrophages (TAMs), and expressed CD47 and calreticulin, the dominant pro-phagocytic molecule. In addition, ATC tissues expressed the immune checkpoint molecules programmed cell death 1 and programmed death ligand 1. Blocking CD47 promoted the phagocytosis of ATC cell lines by macrophages in vitro. Anti-CD47 antibody treatment of ATC xenotransplanted mice increased the frequency of TAMs, enhanced the expression of macrophage activation markers, augmented tumor cell phagocytosis, and suppressed tumor growth. In double-transgenic ATC mice, CD47 was expressed on tumor cells, and blocking CD47 increased TAM frequencies. Conclusions: Targeting CD47 or CD47 in combination with programmed cell death 1 may potentially improve the outcomes of ATC patients and may represent a valuable addition to the current standard of care.

New therapies for anaplastic thyroid cancer.

Anaplastic thyroid cancer is a rare but extremely aggressive type of thyroid cancer. Treatment typically consists of surgery, external beam radiotherapy and cytotoxic chemotherapy. However, available literature suggests only modest survival benefit for cytotoxic chemotherapy. Recent advances have suggested the combination of BRAF and MEK inhibition may have a profound and durable effect on patients with BRAFV600E-mutated anaplastic thyroid cancer, with a response rate of 69%. Other systemic treatments, including immunotherapies, have also shown promising but more limited results. Many clinical trials assessing the efficacy of kinase inhibitors and immunotherapies are currently ongoing.

Combinations of BRAF inhibitor and anti-PD-1/PD-L1 antibody improve survival and tumour immunity in an immunocompetent model of orthotopic murine anaplastic thyroid cancer.

BACKGROUND: Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite aggressive multimodal therapy. ATC has a high prevalence of BRAFV600E mutations and is associated with an immunosuppressive microenvironment; we previously demonstrated that the combination of BRAF inhibitor and checkpoint inhibitor immunotherapy synergistically reduce tumour volume in an immunocompetent mouse model of orthotopic ATC. METHODS: We again utilised our mouse model of ATC to assess the combination of BRAFV600E inhibitor PLX4720 and anti-PD-L1 or anti-PD-1 antibody on survival, and performed immune cell profiling of lymphoid and myeloid-lineage cells during maximal treatment response and tumour regrowth. RESULTS: Combination therapy dramatically improved mouse survival. Maximal tumour reduction was associated with increases in the number and cytotoxicity of CD8+ T cells and NK cells, as well as increases in mostly M1-polarised tumour-associated macrophages (TAM) and decreases in myeloid-derived suppressor-like cells. Regrowth of tumour occurred after 2-3 weeks of ongoing combination therapy, and was most significantly associated with decreased TAMs and a dramatic increase in M2-polarisation. CONCLUSIONS: Combination of PLX4720 and anti-PD-L1/PD-1 antibody dramatically reduced tumour volume, prolonged survival and improved the anti-tumour immune profile in murine ATC. Tumour growth inevitably recurred and demonstrated re-emergence of an immunosuppressive tumour microenvironment.

Dysfunction of natural killer cells mediated by PD-1 and Tim-3 pathway in anaplastic thyroid cancer.

The survival rate of anaplastic thyroid cancer (ATC) remains about 7% to 14%. The natural killer (NK) cells are a critical component of antitumor immunity, and their composition and function in thyroid cancer patients are investigated in this study. In healthy controls and early stage thyroid cancer patients, >90% of circulating NK cells were CD56loCD16hi and fewer than 10% were CD56hiCD16hi/lo. However, the frequency of the CD56hiCD16hi/lo NK subset was significantly higher in more advanced thyroid cancer patients and further increased in ATC patients. Two members of the inhibitory KIR family, CD158a and CD158b, was significantly higher in CD56hiCD16hi/lo NK cells than in CD56loCD16hi NK cells, while NKG2D, an activator of NK cells, was significantly lower in CD56hiCD16hi/lo NK cells than in CD56loCD16hi NK cells. We also found that the CD56hiCD16hi/lo NK cells presented higher PD-1, higher Tim-3, and lower cytotoxicity against the human ATC cell line CAL-62, than the CD56loCD16hi NK cells. The expression of exhaustion markers and reduction in cytotoxicity was further exacerbated in more advanced thyroid cancer patients and in ATC patients. Interestingly, PD-1 and Tim-3 blockade was effective at reinvigorating both the more impaired CD56hiCD16hi/lo NK cells and the less impaired CD56loCD16hi NK cells from ATC patients. Together, our study identified a dysfunction of NK cells in more advanced thyroid cancer patients and ATC patients, and presented actionable targets for future development of immunotherapies in thyroid cancers.

CAR T Therapy Targeting ICAM-1 Eliminates Advanced Human Thyroid Tumors.

Purpose: Poorly differentiated thyroid cancer and anaplastic thyroid cancer (ATC) are rare yet lethal malignancies with limited treatment options. Many malignant tumors, including papillary thyroid cancer (PTC) and ATC, are associated with increased expression of ICAM-1, providing a rationale for utilizing ICAM-1-targeting agents for the treatment of aggressive cancer. We developed a third-generation chimeric antigen receptor (CAR) targeting ICAM-1 to leverage adoptive T-cell therapy as a new treatment modality.Experimental Design: ICAM-1 CAR T cells were applied to multiple malignant and nonmalignant target cells to investigate specific target cell death and "off-tumor" toxicity in vitroIn vivo therapeutic efficacy of ICAM-1 CAR T cells was examined in ATC mouse models established from a cell line and patient-derived tumors that rapidly develop systemic metastases.Results: ICAM-1 CAR T cells demonstrated robust and specific killing of PTC and ATC cell lines in vitro Interestingly, although certain ATC cell lines showed heterogeneous levels of ICAM-1 expression, addition of cytotoxic CAR T cells induced increased ICAM-1 expression such that all cell lines became targetable. In mice with systemic ATC, a single administration of ICAM-1 CAR T cells mediated profound tumor killing that resulted in long-term remission and significantly improved survival. Patient-derived ATC cells overexpressed ICAM-1 and were largely eliminated by autologous ICAM-1 CAR T cells in vitro and in animal models.Conclusions: Our findings are the first demonstration of CAR T therapy against both a metastatic, thyroid cancer cell line and advanced ATC patient-derived tumors that exhibit dramatic therapeutic efficacy and survival benefit in animal studies. Clin Cancer Res; 23(24); 7569-83. ©2017 AACR.